1. Field of Invention
This invention pertains to a designer photosynthetic organism that is specifically designed for production of molecular hydrogen (H2) through photosynthetic water splitting. The various embodiments include (1) targeted genetic insertion of a proton channel into algal thylakoid membrane and (2) novel application of an anaerobic promoter such as a hydrogenase promoter to act as a genetic switch in controlling the expression of a designer proton channel gene. In particular, this invention pertains to a proton-channel designer alga for enhanced photobiological H2 production. Various embodiments solve the following four major problems that currently challenge those in the field of photobiological H2 production: (1) restriction of photosynthetic H2 production by accumulation of a proton gradient, (2) competitive inhibition of photosynthetic H2 production by CO2, (3) requirement of bicarbonate binding at photosystem II (PSII) for efficient photosynthetic activity, and (4) competitive drainage of electrons by O2 in algal H2 production. The application titled “Switchable photosystem-II designer algae for photobiological hydrogen production” and filed on the same day and by the same inventor as this application is hereby incorporated by reference in its entirety.
2. Background
Algal (such as Chlamydomonas reinhardtii, Platymonas subcordiformis, Chlorella fusca, Ankistrodesmus braunii, and Scenedesmus obliquus) photosynthetic hydrogen (H2) production from water has tremendous potential to be a clean and renewable energy resource. However, there are a number of technical issues that must be addressed before algal H2 production can become practical. Currently, there are four proton-gradient-associated physiological problems that challenge researchers and investors in the field of photobiological H2 production are: (1) restriction of photosynthetic H2 production by accumulation of a proton gradient, (2) competitive inhibition of photosynthetic H2 production by CO2, (3) requirement of bicarbonate binding at photosystem II (PSII) for efficient photosynthetic activity, and (4) competitive drainage of electrons by O2 in algal H2 production. In order for the photobiological H2-production technology to work efficiently, all of these four problems must be solved. The various embodiments teach how to generate a switchable proton-channel designer alga to solve these four physiological problems for enhanced photobiological H2 production from water. The following describes the four physiological problems that are solved by various embodiments:
(1). Restriction of photosynthetic H2 production by accumulation of a proton gradient across algal thylakoid membrane. As illustrated in FIG. 1, for each H2 molecule produced photosynthetically, six protons are translocated across the algal thylakoid membrane: two protons are generated by photosystem II (PSII) water splitting at the lumen side and consumed by ferredoxin (Fd)/hydrogenase-catalyzed production of H2 by reduction of protons in the stroma; two protons per pair of electrons flowing from QB site to photosystem I (PSI) are physically taken from the stroma by the reduction of plastoquinone (PQ) to PQH2 at QB site and then released into the lumen with oxidation of PQH2 at the Qo site of the cytochrome b/f (Cyt b/f) complex; and two protons per pair of electrons are transported from the stroma into the lumen by the Q-cycle around the Cyt b/f complex. Since the thylakoid membrane has limited permeability to protons, photosynthetic electron transport will result in accumulation of protons inside the lumen of the thylakoids. In photosynthetic CO2 fixation, this proton gradient across the thylakoid membrane is used by the coupling factor CFoCF1 complex to drive the formation of ATP that is required by the Calvin cycle. However, unlike the Calvin cycle, the Fd/hydrogenase H2 production pathway does not consume ATP. Consequently, under the conditions of H2 photoevolution where the consumption of ATP by the Calvin cycle stops due to the absence of CO2, the CFoCF1-mediated conduction of protons from the lumen to the stroma will quickly become limiting because of the accumulation of ATP and the shutdown of ATP synthase activity. As a result, photosynthetic H2 production quickly results in an increased proton gradient across the thylakoid membrane that has no mechanism for dissipation. The static back-proton gradient seriously impedes the electron transport, thus limiting the rate of H2 production, since electron transport from water through PSII, PQ, Cyt b/f, PC, and PSI to the Fd/hydrogenase pathway is coupled with proton translocation across the thylakoid membrane (FIG. 1). This is one of the reasons that photosynthetic H2 production is saturated at a much lower light intensity than that of photosynthetic CO2 fixation.
(2). Competitive inhibition of photosynthetic H2 production by CO2. As illustrated in FIG. 1, CO2 fixation by the Calvin cycle can compete with the Fd/hydrogenase pathway for photosynthetically generated electrons, resulting in competitive inhibition of H2 production. This inhibition of H2 evolution by CO2 has been characterized in experimental studies at ORNL. The result showed that the algal system is sensitive to CO2 at a concentration as low as 30 ppm. Steady-state H2 photoevolution was inhibited to nearly zero by injection of 58 ppm CO2. Therefore, unless the alga is genetically reengineered to control the Calvin cycle activity, photosynthetic H2 production would require a nearly CO2-free environment.
(3). Requirement for bicarbonate binding at PSII for efficient photosynthetic activity. Bicarbonate (HCO3−) is believed to be an activator of PSII. Experimental studies have demonstrated that addition of HCO3− to HCO3−-depleted samples can result in a 6- to 7-fold stimulation of PSII electron-transport activity. Because CO2 and HCO3− are interchangeable in aqueous medium, removal of CO2 can lead to depletion of HCO3−, thus affecting PSII activity. This presents a dilemma when one tries to reduce the CO2 concentration as required to limit the competitive inhibition of H2 production by CO2.
(4). Competitive drainage of electrons by O2 in algal H2 production. We have recently discovered a new O2 sensitivity in algal H2 production that is distinct from the O2 sensitivity of hydrogenase per se. This O2 sensitivity is apparently linked to the photosynthetic H2 production pathway that is coupled to proton translocation across the thylakoid membrane. Addition of the proton uncoupler FCCP eliminates this mode of O2 inhibition on H2 photoevolution. This inhibition is likely due to the background O2, which apparently serves as a terminal electron acceptor in competition with the H2-production pathway for photosynthetically generated electrons from water splitting. The Rubisco enzyme of the Calvin cycle is known to be also an oxygenase in addition to its carboxylase activity. Therefore, as illustrated in FIG. 1, O2 could act as an electron acceptor through Rubisco at the Calvin cycle with the mechanism known as the photorespiration in competition with the Fd/hydrogenase H2 production pathway for the photosynthetically generated electrons from water. That is, the drainage of electrons by O2 is likely to occur at the point of Rubisco, which is one of the key enzymes of the Calvin cycle. This competitive inhibition of H2 production by O2 is much more of a problem than the traditionally known O2 sensitivity of the hydrogenase per se. Our experimental studies demonstrated that photosynthetic H2 production can be inhibited by an O2 concentration as low as about 500-1000 ppm while the algal hydrogenase is still active at an O2 concentration as high as 5000 ppm. The competitive inhibition of H2 production by O2 is about 10 times more sensitive to O2 than the traditionally known O2 sensitivity of hydrogenase. The drainage of electrons by O2 in competition with the Fd/hydrogenase H2 production pathway is 10 time more sensitive to O2 than the O2 sensitivity of the hydrogenase. Therefore, this is also a serious problem that must be solved in order for the photobiological H2 production technology to work efficiently.
Many of the approaches reported in the field of studies such as the “sulfur-deprivation”-based approach do not solve any of these four major physiological problems that seriously limit the energy efficient and the rate (yield) of algal H2 production. The solar-to-hydrogen energy conversion efficiency of the available algal H2-production technologies including the “sulfur-deprivation”-based approach is typically only less than 0.1%, which is not practical for commercial use. The reason why the solar-to-hydrogen energy conversion efficiency of the photobiological H2-production process is so poor is because the four major physiological problems in algal H2 production described above had not been solved by any of the prior arts in this field. The various embodiments uniquely solve all of these four major problems in algal H2 production through creation of a designer proton-channel transgenic alga with a systematic approach, which improves the solar-to-hydrogen energy conversion efficiency by a factor of more than 10 times in the field of renewable photobiological hydrogen production from water.
The various embodiments are based on new scientific understanding that all of these four problems are somehow associated with the proton gradient across the algal thylakoid membrane in relation to photobiological H2 production. Therefore, it is possible to solve all of these four problems by use of a proton shuttling mechanism such as a proton channel across algal thylakoid membrane. By testing the effect of proton uncouplers such as trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) in algal H2 production, the inventor recently accomplished some new and preliminary understanding that use of proton uncoupler FCCP with a function similar to a proton channel in the thylakoid membrane eliminates both the problem of back-proton accumulation and the newly discovered O2 sensitivity. See, “A new perspective on hydrogen production by photosynthetic water splitting,” by Lee, J. W. and E. Greenbaum, ACS Symposium Series 666, Fuels and Chemicals from Biomass, B. C. Saha and J. Woodward, eds., Chapter 11, pp. 209-222, 1997; “A new oxygen sensitivity and its potential application in photosynthetic H2 production,” by Lee, J. W., and E. Greenbaum, Applied Biochemistry and Biotechnology, Vol. 105-108, pg 303-313, 2003. As demonstrated by the experimental results shown in FIG. 2, addition of 5 μM FCCP produced a dramatic reversal of O2 inhibition on H2 photoevolution. The rate of H2 production rose to about 16 μmol H2/mg Chl.h. This FCCP-stimulated H2 production is clearly photodependent. As soon as the actinic light was turned off, the H2 production stopped. However, most of these chemical proton uncouplers—such as FCCP, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and anilinothiophene—have undesirable side effects, including the acceleration of the deactivation of the water-splitting system Y (ADRY) effect, which can damage the photosynthetic activity in algal cells. See, “ADRY agent-induced cyclic and non-cyclic electron transfer around photosystem II,” by Samuilov, V. D., E. L. Barsky, and A. V. Kitashov, Photosynthesis: from Light to Biosphere, P. Mathis (ed.), Vol. II, 267-270, 1995. As shown in FIG. 2, the FCCP-enhanced photoevolution of H2 can last for more than 4 hours with some decay. This decay is due to the ADRY effect, in which FCCP gradually inhibits photosystem II (PSII) activity by deactivation of the photosynthetic water-splitting complex in the S2 and S3 states. Furthermore, those chemical uncouplers (such as FCCP) are hazardous materials that are environmentally unacceptable for large-scale applications. These challenges and scientific understandings indicate the need of finding a new solution with a polypeptide proton channel that does not have the ADRY effect to solve the four proton-gradient related problems in photobiological H2 production. Consequently, the various embodiments are created with new knowledge and scientific progresses to overcome the roadblocks including the four proton-gradient related problems to photosynthetic H2 production from water.